Background: Gene expression in eukaryotic cells can be governed by histone variants, which replace replication-coupled histones, conferring unique chromatin properties. MacroH2A1 is a histone H2A variant containing a domain highly similar to H2A and a large non-histone (macro) domain. MacroH2A1, in turn, is present in two alternatively exon-spliced isoforms: macroH2A1.1 and macroH2A1.2, which regulate cell plasticity and proliferation in a remarkably distinct manner. The N-terminal and the C-terminal tails of H2A histones stem from the nucleosome core structure and can be target sites for several post-translational modifications (PTMs). MacroH2A1.1 and macroH2A1.2 isoforms differ only in a few amino acids and their ability to bind NAD-derived metabolites, a property allegedly conferring their different functions in vivo. Some of the modifications on the macroH2A1 variant have been identified, such as phosphorylation (T129, S138) and methylation (K18, K123, K239). However, no study to our knowledge has analyzed extensively, and in parallel, the PTM pattern of macroH2A1.1 and macroH2A1.2 in the same experimental setting, which could facilitate the understanding of their distinct biological functions in health and disease. Methods: We used a mass spectrometry-based approach to identify the sites for phosphorylation, acetylation, and methylation in green fluorescent protein (GFP)-tagged macroH2A1.1 and macroH2A1.2 expressed in human hepatoma cells. The impact of selected PTMs on macroH2A1.1 and macroH2A1.2 structure and function are demonstrated using computational analyses. Results: We identified K7 as a new acetylation site in both macroH2A1 isoforms. Quantitative comparison of histone marks between the two isoforms revealed significant differences in the levels of phosphorylated T129 and S170. Our computational analysis provided evidence that the phosphorylation status in the intrinsically disordered linker region in macroH2A1 isoforms might represent a key regulatory element contributing to their distinct biological responses. Conclusions: Taken together, our results report different PTMs on the two macroH2A1 splicing isoforms as responsible for their distinct features and distribution in the cell.

Department of Biology Faculty of Medicine Masaryk University Kamenice 753 5 62500 Brno Czech Republic

Department of Molecular Medicine Sapienza University of Rome Viale Regina Elena 291 00161 Rome Italy

Department of Translational Stem Cell Biology Medical University of Varna 9002 Varna Bulgaria

International Clinical Research Center St'Anne University Hospital Pekařská 53 65691 Brno Czech Republic

IRCCS Casa Sollievo della Sofferenza Bioinformatics Unit Viale Cappuccini 1 71013 San Giovanni Rotondo Italy

Laboratory of Functional Genomics and Proteomics National Centre for Biomolecular Research Faculty of Science Masaryk University Kamenice 753 5 62500 Brno Czech Republic

Mendel Centre for Plant Genomics and Proteomics Central European Institute of Technology Masaryk University Kamenice 753 5 62500 Brno Czech Republic

Zobrazit více v PubMed

Buschbeck M., Hake S.B. Variants of core histones and their roles in cell fate decisions, development and cancer. Nat. Rev. Mol. Cell Biol. 2017;18:299–314. doi: 10.1038/nrm.2016.166. PubMed DOI

Lo Re O., Vinciguerra M. Histone MacroH2A1: A Chromatin Point of Intersection between Fasting, Senescence and Cellular Regeneration. Genes. 2017;8:367. doi: 10.3390/genes8120367. PubMed DOI PMC

Pehrson J.R., Fried V.A. MacroH2A, a core histone containing a large nonhistone region. Science. 1992;257:1398–1400. doi: 10.1126/science.1529340. PubMed DOI

Buzova D., Maugeri A., Liguori A., Napodano C., Lo Re O., Oben J., Alisi A., Gasbarrini A., Grieco A., Cerveny J., et al. Circulating histone signature of human lean metabolic-associated fatty liver disease (MAFLD) Clin. Epigenetics. 2020;12:126. doi: 10.1186/s13148-020-00917-2. PubMed DOI PMC

Bruno M., Flaus A., Stockdale C., Rencurel C., Ferreira H., Owen-Hughes T. Histone H2A/H2B dimer exchange by ATP-dependent chromatin remodeling activities. Mol. Cell. 2003;12:1599–1606. doi: 10.1016/S1097-2765(03)00499-4. PubMed DOI PMC

Huang H., Sabari B.R., Garcia B.A., Allis C.D., Zhao Y. SnapShot: Histone modifications. Cell. 2014;159:458–458 e451. doi: 10.1016/j.cell.2014.09.037. PubMed DOI PMC

Corujo D., Buschbeck M. Post-Translational Modifications of H2A Histone Variants and Their Role in Cancer. Cancers. 2018;10:59. doi: 10.3390/cancers10030059. PubMed DOI PMC

Sporn J.C., Jung B. Differential regulation and predictive potential of MacroH2A1 isoforms in colon cancer. Am. J. Pathol. 2012;180:2516–2526. doi: 10.1016/j.ajpath.2012.02.027. PubMed DOI PMC

Sporn J.C., Kustatscher G., Hothorn T., Collado M., Serrano M., Muley T., Schnabel P., Ladurner A.G. Histone macroH2A isoforms predict the risk of lung cancer recurrence. Oncogene. 2009;28:3423–3428. doi: 10.1038/onc.2009.26. PubMed DOI

Novikov L., Park J.W., Chen H., Klerman H., Jalloh A.S., Gamble M.J. QKI-mediated alternative splicing of the histone variant MacroH2A1 regulates cancer cell proliferation. Mol. Cell. Biol. 2011;31:4244–4255. doi: 10.1128/MCB.05244-11. PubMed DOI PMC

Hurtado-Bages S., Guberovic I., Buschbeck M. The MacroH2A1.1—PARP1 Axis at the Intersection Between Stress Response and Metabolism. Front. Genet. 2018;9:417. doi: 10.3389/fgene.2018.00417. PubMed DOI PMC

Kustatscher G., Hothorn M., Pugieux C., Scheffzek K., Ladurner A.G. Splicing regulates NAD metabolite binding to histone macroH2A. Nat. Struct. Mol. Biol. 2005;12:624–625. doi: 10.1038/nsmb956. PubMed DOI

Marjanovic M.P., Hurtado-Bages S., Lassi M., Valero V., Malinverni R., Delage H., Navarro M., Corujo D., Guberovic I., Douet J., et al. MacroH2A1.1 regulates mitochondrial respiration by limiting nuclear NAD+ consumption. Nat. Struct. Mol. Biol. 2017;24:902–910. doi: 10.1038/nsmb.3481. PubMed DOI PMC

Chu F., Nusinow D.A., Chalkley R.J., Plath K., Panning B., Burlingame A.L. Mapping post-translational modifications of the histone variant MacroH2A1 using tandem mass spectrometry. Mol. Cell. Proteom. 2006;5:194–203. doi: 10.1074/mcp.M500285-MCP200. PubMed DOI

Jueliger S., Lyons J., Cannito S., Pata I., Pata P., Shkolnaya M., Lo Re O., Peyrou M., Villarroya F., Pazienza V., et al. Efficacy and epigenetic interactions of novel DNA hypomethylating agent guadecitabine (SGI-110) in preclinical models of hepatocellular carcinoma. Epigenetics. 2016;11:709–720. doi: 10.1080/15592294.2016.1214781. PubMed DOI PMC

Lo Re O., Fusilli C., Rappa F., Van Haele M., Douet J., Pindjakova J., Rocha S.W., Pata I., Valcikova B., Uldrijan S., et al. Induction of cancer cell stemness by depletion of macrohistone H2A1 in hepatocellular carcinoma. Hepatology. 2018;67:636–650. doi: 10.1002/hep.29519. PubMed DOI

Borghesan M., Fusilli C., Rappa F., Panebianco C., Rizzo G., Oben J.A., Mazzoccoli G., Faulkes C., Pata I., Agodi A., et al. DNA Hypomethylation and Histone Variant macroH2A1 Synergistically Attenuate Chemotherapy-Induced Senescence to Promote Hepatocellular Carcinoma Progression. Cancer Res. 2016;76:594–606. doi: 10.1158/0008-5472.CAN-15-1336. PubMed DOI PMC

Legartova S., Lochmanova G., Zdrahal Z., Kozubek S., Sponer J., Krepl M., Pokorna P., Bartova E. DNA Damage Changes Distribution Pattern and Levels of HP1 Protein Isoforms in the Nucleolus and Increases Phosphorylation of HP1beta-Ser88. Cells. 2019;8:1097. doi: 10.3390/cells8091097. PubMed DOI PMC

Altschul S.F., Gish W., Miller W., Myers E.W., Lipman D.J. Basic local alignment search tool. J. Mol. Biol. 1990;215:403–410. doi: 10.1016/S0022-2836(05)80360-2. PubMed DOI

Berman H.M., Westbrook J., Feng Z., Gilliland G., Bhat T.N., Weissig H., Shindyalov I.N., Bourne P.E. The Protein Data Bank. Nucleic Acids Res. 2000;28:235–242. doi: 10.1093/nar/28.1.235. PubMed DOI PMC

Pettersen E.F., Goddard T.D., Huang C.C., Couch G.S., Greenblatt D.M., Meng E.C., Ferrin T.E. UCSF Chimera--a visualization system for exploratory research and analysis. J. Comput. Chem. 2004;25:1605–1612. doi: 10.1002/jcc.20084. PubMed DOI

Parca L., Ariano B., Cabibbo A., Paoletti M., Tamburrini A., Palmeri A., Ausiello G., Helmer-Citterich M. Kinome-wide identification of phosphorylation networks in eukaryotic proteomes. Bioinformatics. 2019;35:372–379. doi: 10.1093/bioinformatics/bty545. PubMed DOI PMC

Parca L., Ferre F., Ausiello G., Helmer-Citterich M. Nucleos: A web server for the identification of nucleotide-binding sites in protein structures. Nucleic Acids Res. 2013;41:W281–W285. doi: 10.1093/nar/gkt390. PubMed DOI PMC

Parca L., Gherardini P.F., Truglio M., Mangone I., Ferre F., Helmer-Citterich M., Ausiello G. Identification of nucleotide-binding sites in protein structures: A novel approach based on nucleotide modularity. PLoS ONE. 2012;7:e50240. doi: 10.1371/journal.pone.0050240. PubMed DOI PMC

Chakravarthy S., Gundimella S.K., Caron C., Perche P.Y., Pehrson J.R., Khochbin S., Luger K. Structural characterization of the histone variant macroH2A. Mol. Cell. Biol. 2005;25:7616–7624. doi: 10.1128/MCB.25.17.7616-7624.2005. PubMed DOI PMC

Timinszky G., Till S., Hassa P.O., Hothorn M., Kustatscher G., Nijmeijer B., Colombelli J., Altmeyer M., Stelzer E.H., Scheffzek K., et al. A macrodomain-containing histone rearranges chromatin upon sensing PARP1 activation. Nat. Struct. Mol. Biol. 2009;16:923–929. doi: 10.1038/nsmb.1664. PubMed DOI

Ruiz P.D., Gamble M.J. MacroH2A1 chromatin specification requires its docking domain and acetylation of H2B lysine 20. Nat. Commun. 2018;9:5143. doi: 10.1038/s41467-018-07189-8. PubMed DOI PMC

Lo Re O., Douet J., Buschbeck M., Fusilli C., Pazienza V., Panebianco C., Castracani C.C., Mazza T., Li Volti G., Vinciguerra M. Histone variant macroH2A1 rewires carbohydrate and lipid metabolism of hepatocellular carcinoma cells towards cancer stem cells. Epigenetics. 2018;13:829–845. doi: 10.1080/15592294.2018.1514239. PubMed DOI PMC

Seet B.T., Dikic I., Zhou M.M., Pawson T. Reading protein modifications with interaction domains. Nat. Rev. Mol. Cell Biol. 2006;7:473–483. doi: 10.1038/nrm1960. PubMed DOI

Hornbeck P.V., Zhang B., Murray B., Kornhauser J.M., Latham V., Skrzypek E. PhosphoSitePlus, 2014: Mutations, PTMs and recalibrations. Nucleic Acids Res. 2015;43:D512–D520. doi: 10.1093/nar/gku1267. PubMed DOI PMC

Zink L.M., Hake S.B. Histone variants: Nuclear function and disease. Curr. Opin. Genet. Dev. 2016;37:82–89. doi: 10.1016/j.gde.2015.12.002. PubMed DOI

Pehrson J.R., Fuji R.N. Evolutionary conservation of histone macroH2A subtypes and domains. Nucleic Acids Res. 1998;26:2837–2842. doi: 10.1093/nar/26.12.2837. PubMed DOI PMC

Rappa F., Greco A., Podrini C., Cappello F., Foti M., Bourgoin L., Peyrou M., Marino A., Scibetta N., Williams R., et al. Immunopositivity for histone macroH2A1 isoforms marks steatosis-associated hepatocellular carcinoma. PLoS ONE. 2013;8:e54458. doi: 10.1371/annotation/b456329c-02fa-4055-afb8-2090cec17da6. PubMed DOI PMC

Cantarino N., Douet J., Buschbeck M. MacroH2A—An epigenetic regulator of cancer. Cancer Lett. 2013;336:247–252. doi: 10.1016/j.canlet.2013.03.022. PubMed DOI

Angelov D., Molla A., Perche P.Y., Hans F., Cote J., Khochbin S., Bouvet P., Dimitrov S. The histone variant macroH2A interferes with transcription factor binding and SWI/SNF nucleosome remodeling. Mol. Cell. 2003;11:1033–1041. doi: 10.1016/S1097-2765(03)00100-X. PubMed DOI

Chevanne M., Zampieri M., Caldini R., Rizzo A., Ciccarone F., Catizone A., D’Angelo C., Guastafierro T., Biroccio A., Reale A., et al. Inhibition of PARP activity by PJ-34 leads to growth impairment and cell death associated with aberrant mitotic pattern and nucleolar actin accumulation in M14 melanoma cell line. J. Cell. Physiol. 2010;222:401–410. doi: 10.1002/jcp.21964. PubMed DOI

Carbone M., Rossi M.N., Cavaldesi M., Notari A., Amati P., Maione R. Poly(ADP-ribosyl)ation is implicated in the G0-G1 transition of resting cells. Oncogene. 2008;27:6083–6092. doi: 10.1038/onc.2008.221. PubMed DOI

Bernstein E., Muratore-Schroeder T.L., Diaz R.L., Chow J.C., Changolkar L.N., Shabanowitz J., Heard E., Pehrson J.R., Hunt D.F., Allis C.D. A phosphorylated subpopulation of the histone variant macroH2A1 is excluded from the inactive X chromosome and enriched during mitosis. Proc. Natl. Acad. Sci. USA. 2008;105:1533–1538. doi: 10.1073/pnas.0711632105. PubMed DOI PMC

Maiolica A., de Medina-Redondo M., Schoof E.M., Chaikuad A., Villa F., Gatti M., Jeganathan S., Lou H.J., Novy K., Hauri S., et al. Modulation of the chromatin phosphoproteome by the Haspin protein kinase. Mol. Cell. Proteomics. 2014;13:1724–1740. doi: 10.1074/mcp.M113.034819. PubMed DOI PMC

Muthurajan U.M., McBryant S.J., Lu X., Hansen J.C., Luger K. The linker region of macroH2A promotes self-association of nucleosomal arrays. J. Biol. Chem. 2011;286:23852–23864. doi: 10.1074/jbc.M111.244871. PubMed DOI PMC

Chakravarthy S., Patel A., Bowman G.D. The basic linker of macroH2A stabilizes DNA at the entry/exit site of the nucleosome. Nucleic Acids Res. 2012;40:8285–8295. doi: 10.1093/nar/gks645. PubMed DOI PMC

Kozlowski M., Corujo D., Hothorn M., Guberovic I., Mandemaker I.K., Blessing C., Sporn J., Gutierrez-Triana A., Smith R., Portmann T., et al. MacroH2A histone variants limit chromatin plasticity through two distinct mechanisms. EMBO Rep. 2018;19:e44445. doi: 10.15252/embr.201744445. PubMed DOI PMC

Histone Variant macroH2A1.1 Enhances Nonhomologous End Joining-dependent DNA Double-strand-break Repair and Reprogramming Efficiency of Human iPSCs